GB2122197A - Antiviral guanine derivatives - Google Patents

Abstract

Antiviral compounds have the formula <IMAGE> wherein R1 is hydrogen, hydroxy or mercapto. The compounds and their physiologically acceptable salts and optical isomers and pharmaceutical compositions containing them are prepared by various methods.

Description

1

GB 2 122 197 A 1

SPECIFICATION

Novel derivatives of guanine II

Field of the invention

The present invention relates to novel derivatives of guanine, methods for their preparation, novel 5 pharmaceutical compositions and use of said compounds in the treatment of virus infections, such as 5 herpes virus infections, which can cause various diseases in an animal or human host, including both common infections and neoplastic diseases, i.e. cancer.

Background of the invention

The effects of viruses on bodily functions is the end result of changes occurring at the cellular and 1 o subcellular levels. The pathogenic changes at the cellular level are different for different combinations 10 of viruses and host cells. While some viruses cause a general destruction (killing) of certain cells, other may transform cells to a neoplastic state.

Important common viral infections are herpes dermatitis (including herpes labialis), herpes keratitis, herpes genitalis, herpes zoster, herpes encephalitis, infectious mononucleosis and 15 cytomegalovirus infections all of which are caused by viruses belonging to the herpes virus group. 15 Other important viral diseases are influenza A and B which are caused by influenza A and B virus respectively. Another important common viral disease is viral hepatitis and especially hepatitis B virus infections are widely spread. Effective and selective antiviral agents are needed for the treatment of these diseases as well as for other diseases caused by viruses.

20 Several different viruses of both DNA and RNA type have been shown to cause tumors in animals. 20

The effect of cancerogenic chemicals can on animals result in activation of latent tumor viruses. It is possible that tumor viruses are involved in human tumors. The most likely human cases known today are leucemias, sarcomas, breast carcinomas, Burkitt lymphomas, nasopharyngeal carcinomas and cervical cancers where RNA tumor viruses and herpes viruses are indicated. This makes the search for 25 selective inhibitors of tumororgenic viruses and their functions an important undertaking in the efforts to 25 treat cancer.

Prior art

The compound 9-(4-hydroxybutyl)-guanine is disclosed in Chem. Pharm. Bull. 17 (1969) 1268— 1270 and in Agr. Biol. Chem., 37 (1973) 2037—2043. However, no antiviral or other 30 pharmacological activity has been disclosed for said compound. 30

US 4 199 574 discloses a broad class of substituted purines of the formula

,1

—CH-X-CH - CH-R5 lK I, I,'

R R3 R4

wherein X is oxygen or sulphur; R1 is hydrogen, halogen, hydroxy, alkoxy, azide, thio, alkylthio, amino, alkylamino, or dialkylamino; R2 is hydrogen, halogen, alkylthio, acylamino, amino or azide; R3 is 35 hydrogen, straight or branch chain or cyclic alkyl, hydroxyalkyl, benzyloxyalkyl, or phenyl; R4 is 35

hydrogen, hydroxy or alkyl; R5 is hydrogen, hydroxy, amino, alkyl, hydroxyalkyl, benzyloxy, benzoyloxy, benzoyloxymethyl, sulphamoyloxy, phosphate carboxypropionyloxy, straight chain or cyclic acyloxy having from 1 to 8 carbon atoms e.g., acetoxy or substituted carbamoyl group of formula NHCO-Z wherein Z is alkyl, aryl or aralkyl optionally substituted by one or more of sulphonyl, amino, carbamoyl 40 or halogen; R6 is hydrogen or alkyl, provided that when X is oxygen and R2, R3, R4 and R6 are hydrogen, 40 R1 is not amino or methylamino when R5 is hydrogen or hydroxy. These compounds are asserted to possess antiviral activity against various classes of DNA and RNA viruses. 9-(2-HydroxyethoxymethyDguanine and 2-amino-9-(2-hydroxyethoxymethyl)adenine are mentioned as examples of especially active compounds.

45 Disclosure of invention 45

The present invention relates to the novel antiviral compound of the formula h2n n ch, - c - ch - chgoh

2

GB 2 122 197 A 2

wherein R, is hydrogen, hydroxy or mercapto and physiologically acceptable salts or optical isomers thereof.

It has been found that such compound exerts an antiviral effect and inhibits certain viral functions including tumorogenic functions and the multiplication of viruses.

5 The invention thus provides a compound, a physiologically acceptable salts thereof, which 5

compounds are useful in therapeutic and/or prophylactic treatment of viral diseases and which may be useful in therapeutic and/or prophylactic treatment of cancer caused by viruses.

An effective selective antiviral agent with acceptable side effects should have a selective inhibiting effect on a specific viral function of the virus to be combated. It is, therefore, one object of the

10 present invention to provide a novel method for combating virus infections using an antiviral agent 10

which exerts a selective inhibiting effect on viral functions but which exerts only a negligible inhibiting effect on functions of the host cells.

The invention also relates to novel pharmaceutical compositions containing the antiviral agents.

Although the present invention relates broadly to a novel method for combating virus infections

15 in animals and man, and compounds to be used at such treatment, it will be particularly useful in the 15 treatment of herpes virus infections.

An especially important area of use for the compounds of the present invention is in the treatment of herpesvirus infections. Among the herpesviruses may be mentioned Herpes simplex type 1 and 2, varicella (Herpes zoster), virus causing infections mononucleosis (i.e. Epstein-Barr virus) and

20 cytomegalovirus. Important diseases caused by herpesviruses are herpes dermatitis (including herpes 20 labialis), herpes genitalis, herpes keratitis, herpes encephalitis and herpes zoster.

Another possible area of use for the compounds of the present invention are in the treatment of cancer and tumors, particularly those caused by viruses. This effect may be obtained in different ways,

i.e. by inhibiting the transformation of virus-infected cells to a neoplastic state, by inhibiting the spread

25 of viruses from transformed cells to other normal cells and by arresting the growth of virus transformed 25 cells.

A further area of use for the compounds of the present invention is in the inhibition of transformed cells due to the presence in these cells of specific herpes virus enzymes like thymidine kinase.

30 Possible areas of use for the compounds of the present invention with respect to cancer 30

chemotherapy are treatment of leucemias, lymphomas including Burkitt lymphomas and Hodgkin's disease, sarcomas, breast carcinoma, nasopharyngeal carcinomas and cervical cancers in which viruses are indicated. Other possible areas of use for the compounds of the present invention with respect to cancer chemotherapy are treatment of multiple myeloma and cancer of the lungs (and

35 bronchus), the stomach, the liver, the colon, the bladder, the lips, the bones, the kidneys, the ovary, the 35 prostate, the pancreas, the skin (melanoma), the rectum, the salivary glands, the mouth, the esophagus, the testis, the brain (and cranial meninges), the thyroid gland, the gallbladder (and ducts), the nose, the larynx, connective tissues, the penis, the vulvas, the vagina, the corpus uteri and the tongue.

40 The invention furthermore provides 40

A. A method for the treatment of diseases caused by viruses in animals including man,

comprising administering to an animal so infected a therapeutically effective amount of a compound of the formula I or a physiologically acceptable salt thereof.

B. A method for inhibiting the multiplication of virus, in particular herpesviruses, in animals

45 including man, by administering to an animal in need of such treatment a compound of the formula I or 45 a physiologically acceptable salt thereof in an amount sufficient for inhibiting said multiplication.

C. A method for the treatment of virus-induced neoplastic diseases in animals including man, by inhibiting the growth of cells expressing viral functions, characterized by administering to an animal so infected a therapeutically effective amount of a compound of the formula I or a.physiologically

50 acceptable salt thereof. 50

D. A method for inhibiting the growth of virus-transformed cells in animals including man,

characterized by administering to an animal in need of such treatment a compound of the formula I or a physiologically acceptable salt thereof in an amount sufficient for inhibiting said growth.

E. A method for the treatment of virus-induced neoplastic diseases in animals including man, by

55 inhibiting the multiplication of tumor viruses, characterized by administering to an animal in need of 55 such treatment a compound of the formula I or a physiologically acceptable salt thereof in an amount sufficient for inhibiting such multiplication.

F. A method for the treatment of neoplastic diseases in animals including man, characterized by administering to an animal a therapeutically effective amount of a compound of the formula I or a

60 physiologically acceptable salt thereof. 60

The invention also relates to the use of a compound of the formula I or a physiologically acceptable salt thereof, in each of the above given methods A, B, C, D, E and F.

As stated previously the compound of the present invention has the formula

GB 2 122 197 A

h2n n

I

- ch - ch9oh i 2

Ri wherein R1 is hydrogen, hydroxy or mercapto including physiologically acceptable salts and optical isomers thereof.

The provisos in the definition for the group R, above mean that only the following three specific 5 compounds, including salts and optical isomers thereof, constitute part of the present invention: 5

9-(2-oxo-4-hydroxybuty!)guanine 9-(2-oxo-3,4-dihydroxybutyl)guanine 9-(2-oxo-3-mercapto-4-hydroxybutyl)guanine

Two of the compounds of the formula I contain one asymmetric center. Accordingly, they exist in 10 two optical forms, respectively, and all such forms constitute a further aspect of the invention. ®

Methods of preparation

The compounds of the invention may be obtained by one of the following methods A—D constituting a further aspect of the invention.

A. Condensing an acyclic side chain, where the functional groups may optionally be protected, to 15 the N-9 position of a guanine derivative, followed by removal of the protecting groups, through one or 15 more chemical reactions.

CH20Rg wherein X is a group such as chlorine, bromine, iodine or a group 0S02R9 where Rg is alkyl containing •j—8 carbon atoms, fluorinated alkyl containing 1—8 carbon atoms, such as trifluoromethyl, aryl or

4

GB 2 122 197 A 4

arylalkyl such as benzyl. Y is hydrogen or a quaternary ammonium ion such as for example tetrabutylammonium. R2 and R3 are together double bonded oxygen, i.e. CR2R3 is carbonyl, or alkoxy groups, forming open or cyclic ketais. R4 is hydrogen or a hydroxyl protecting group of which a great variety are known to those skilled in the art and are described for example in "Protective Groups in 5 Organic Chemistry" (T. W. Greene, Wiley 1981), "Methoden der Organischen Chemie" (Houben-Weyl) 5 Vol. Vl/lb, or in "Comprehensive Organic Chemistry" (D. H. R. Barton and W. D. Ollis eds, 1979) Vol. a, p. 623—629. Just some examples of R4 are acyl groups such as acetyl or benzoyl, alkoxycarbonyl or aryloxycarbonyl groups, silyl groups such as for example tert.-butyl dimethylsilyl, arylalkyl such as benzyl and triarylmethyl or SOzRg where Rg is as defined above.

10 Rs is R, as defined above, or 0R4 or SR4 where R4 is defined above. 10

R4 and R5 may together form an epoxide when R, is OH and R4 and Rs may additionally form a cyclic derivative such as for example a carbonate ester, carbonate thioester or the corresponding ortho acid cyclic derivatives or cyclic acetal type compounds.

R6 is hydroxyl, chlorine, bromine, iodine, thio, thioester, S02R9 where Rg is as defined above; or an 15 oxygen derivative 0Rn where Rn is alkyl, arylalkyl such as benzyl, substituted silyl, phosphoryl diester, 15 phosphinothioyl, or S02Rg where R9 is as defined above. R7 and R8 are the same or different and are hydrogen or R10, where R10 is an amine protecting group known to those skilled in the art and described for example in "Protective Groups in Organic Chemistry" (T. W. Greene, Wiley 1981), "Methoden der Organischen Chemie" (Houben-Weyl) Vol. XI/1 p. 1005 cont'd or in "Comprehensive Organic 20 Chemistry" (D. H. R. Barton and W. D. Ollis eds, 1979) Vol. 2, p. 49—52. Some examples of R10 are 20 acyl groups, alkoxycarbonyl or aryloxycarbonyl groups or silyl groups.

The condensation is preferably conducted in an organic solvent such as for example dimethylformamide, ethanol, acetonitrile or dichloromethane, at a temperature of between 0°C and 100°C for 1 hour to 3 days in the presence of a base (when Y is H) such as for example potassium 25 carbonate. 25

After condensation, the compounds are hydrolyzed at 0—100°Cfor 1—24 hours with acid or base such as for example acetic acid, hydrochloric acid (1—35%) in water, sodium hydroxide (1 — 20%) in water, ammonia (1—25%), in water or methanol, or hydrogenated with hydrogen gas in an organic solvent such as for example ethanol or dimethylformamide over a metal catalyst for 1—24 30 hours at a pressure of 0.1—5 MPa. 30

B1. By substitution reactions on a guanidine N-9 derivatized 4-carbon side-chain.

ch2or4

oh ch2or4

R2 R3 R1

R2, R3, R4, R7 and R8 are as defined above. R12 is iodine or 0S02Rg where Rg is as defined above.

The substitution reactions are performed in an organic solvents such as dimethylformamide, 35 ethanol, acetonitrile or dichloromethane at a temperature of between 0°C and 100°Cfor 1—24 hours 35 and the substituting reagent will be water, ammonia or hydrogen sulfide or their respective ions or ion pairs.

When R4, R7 and Rs are not hydrogen then in a following reaction they are removed according to method A.

40 B2. By reduction of an ester group to an alcohol. 40

5

GB 2 122 197 A 5

ch2dh

R2, R3, R5, R6, R7, R8 and Rg are as defined above. The reduction may be performed with hydrogen gas or hydrogen generated in situ, with a metal as catalyst or with a hydride reducing agent in an organic solvent.

5 C1. Pyrimidine ring closure to the guanine base of a substituted imidazole derivative. 5

d wherein R2, R3, R4 and Rs are as defined above, Z is NH2 or alkoxy i.e. COZ is an amide or ester group and R13 is NH2 or guanidine. The ring closure may be performed by known methods, the principles of which are given for example in "Comprehensive Organic Chemistry" p. 505—508 (1979, Vol. 4, 10 D. H. R. Barton and W. D. Ollis eds). 10

The ring closure is performed in an organic solvent at a temperature from 50° to 250° with or without the addition of a reagent such as for example guanidine. When R4 is not H and R5 is not Rv then the side chain protecting groups are removed in a following reaction step according to method A. C2. Imidazole ring closure, to the guanine base, of a substituted pyrimidine derivative.

6

GB 2 122 197 A 6

-> xJ

h2n

/ \ I

ch

I

Rr

■ch20r4

wherein R,

as formic amide (—NH-

R3, R4 and R5 are as defined above. R

14 is nitroso, nitro, amino or an amino derivative such CHO) or amino ortho ester (e.g.

OC2H5

-NH—CH

10

\

0C2H5

The ring closure may be performed by known methods, the principles of which are given for example in "Comprehensive Organic Chemistry" p. 499—504 (1979, Vol. 4, D. H. R. Barton and W. D."Ollis eds).

The ring closure may be performed in an organic solvent such as for example formic acid, formamide, orthoformate ester at a temperature from 50 to 250°C for 1/2 hour to 10 hours. When R14 is nitroso or nitro, these groups first have to be reduced to amino groups by known methods. When R4 is not H and R5 is not Rv then the side chain protecting groups are removed in a following reaction step according to method A.

D. Substitution in the pyrimidine ring of a purine to the formation of a guanine ring.

10

ch-

/\ l ch20r4

R2 R3

7

5

10

15

20

25

30

35

40

45

50

55

GB 2 122 197 A 7

wherein R2, R3, R4 and Rs are as defined above. Hal is fluoro, chloro, bromo or iodo and R15 is hydroxyl or amino. The halogen atoms are substituted by ammonia in an organic solvent such as methanol, from normal to higher pressure at room temperature to 100°C for 1 to 25 hours or by an azide ion followed by hydrogenation by known methods. When R1S is amino the amino group can be substituted to a hydroxyl function by selective diazotization with nitrite in a solvent such as acetic acid at a temperature from 0°C to 50°C for 1—24 hours, or enzymatically with adenosinedeaminase in water at a pH from 6 to 9 for 1 to 48 hours. When R4 is not hydrogen and R5 is not R,, then the side chain protecting groups are removed in a following reaction step according to method A.

The described methods A—D may be used to give enantiomeric mixtures, or in appropriate cases a single enantiomeric isomer. Additionally a single enantiomeric isomer may be obtained from the enantiomeric mixtures by methods known perse.

The starting materials in the above methods A—D are either known compounds or can be prepared by methods known to those skilled in the art.

Salts

Physiologically acceptable salts of compounds of the invention are prepared by methods known in the art. The salts are novel compounds and comprise a further aspect of the invention. Metal salts can be prepared by treating a metal hydroxide with a compound of the invention. Examples of metal salts which can be prepared in this way are salts containing Li, Na and K. A less soluble metal salt can be precipitated from a solution of a more soluble salt by addition of a suitable metal compound. Acid salts can be prepared by treating a compound of the invention with an acid as HCI, HBr, H2S04, or an organic sulphonic acid.

Pharmaceutical compositions

Pharmaceutical compositions of the compounds of the" invention constitute a further aspect of the invention.

In clinical practice the compounds of the invention may be administered locally or systemically. They will normally be administered topically, orally, intranasally, by injection or by inhalation in the form of a pharmaceutical composition comprising the active ingredient in the form of the original compound or optionally in the form of a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such compositions comprise a further aspect of the invention. The compound may also be used without carrier material. As examples of pharmaceutical compositions may be mentioned tablets, drops, such as nasal drops, eye drops, preparations for topical application such as ointments, jellies, creams and suspensions, aerosols for inhalation, nasal spray, liposomes etc. Usually the active substance will comprise between 0.01 and 99, or between 0.1 and 99% by weight of the composition, for example between 0.5 and 20% for compositions intended for injection and between 0.1 and 50% for compositions intended for oral administration.

The compositions are preferably in dosage unit form. Further, they are preferably provided in sterilized form.

To produce pharmaceutical compositions in the form of dosage units for oral application containing a compound of the invention the active ingredient may be mixed with a solid, pulverulent carrier, for example lactose, saccharose, sorbitol, mannitol, a starch such as potato starch, corn starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a CarbowaxR or other polyethylene glycol waxes and compressed to form tablets or cores for dragees. If dragees are required, the cores may be coated for example with concentrated sugar solutions which may contain gum arabic, talc and/or titanium dioxide, or alternatively with a film forming agent dissolved in easily volatile organic solvents or mixtures or organic solvents. Dyestuffs can be added to these coatings, for example, to distinguish between different contents of active substance. For the preparation of soft gelatine capsules consisting of gelatine and, for example, glycerol and a plasticizer, or similar closed capsules, the active substances may be admixed with a CarbowaxR or a suitable oil as e.g. sesame oil, olive oil, or arachis oil. Hard gelatine capsules may contain granulates of the active substance with solid, pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches (for example potato starch, corn starch or amylopectin), cellulose derivatives or gelatine, and may also include magnesium stearate or stearic acid as lubricants.

By using several layers of the active drug, separated by slowly dissolving coatings sustained release tablets are obtained. Another way of preparing sustained release tablets is to divide the dose of the active drug into granules with coatings of different thicknesses and compress the granules into tablets together with the carrier substance. The active substance can also be incorporated in slowly dissolving tablets made for instance of fat and wax substances or evenly distributed in a tablet of an insoluble substance such as a physiologically inert plastic substance.

Liquid compositions for oral application may be in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1% to 20% by weight of active substance, sugar and a

5

10

15

20

25

30

35

40

45

50

55

60

8

GB 2 122 197 A 8

mixture of ethanol, water, glycerol, propylene glycol and optionally aroma, saccharine and/or carboxymethylcellulose as a dispersing agent.

For parenteral application by injection compositions may comprise an aqueous solution of the active drug or a physiologically acceptable salt thereof, desirably in a concentration of 0.05—10%, 5 and optionally also a stabilizing agent and/or buffer substances in aqueous solution. Dosage units of the 5 solution may advantageously be enclosed in ampoules.

For topical application, especially for the treatment of herpesvirus infections on skin, genitals and in mouth and eyes the compositions are suitably in the form of a solution, ointment, gel, suspension,

cream or the like. The amount of active substance may vary, for example between 0.05—20% by 10 weight of the active substance. Such compositions for topical application may be prepared in known 10 manner by mixing the active substance with known carrier materials such as isopropanol, glycerol,

paraffin, stearyl alcohol, polyethylene glycol, etc. The pharmaceutically acceptable carrier may also include a known chemical absorption powder. Examples of absorption promoters are e.g.

dimethylacetamide (US 3,472,931), trichloroethanol ortrifluoroethanol (US 3,891,757), certain 1 5 alcohols and mixtures thereof (GB 1,001,949). 1 5

The dosage at which the active ingredients are administered may vary within a wide range and will depend on various factors such as for example the severity of the infection, the age of the patient, etc., and may have to be individually adjusted. As a possible range for the amount of the compounds of the invention which may be administered per day may be mentioned from about 0.1 mg to about 2000 20 mg or from about 1 mg to about 2000 mg, or preferably from 1 mg to about 2000 mg for topical 20

administration, from 50 mg to about 2000 mg or from 100 to about 1000 mg for oral administration and from 10 mg to about 2000 mg or from 50 to about 500 mg for injection.

In severe cases it may be necessary to increase these doses 5-fold to 10-fold. In less severe cases it may be sufficient to use up to 500 or 1000 mg.

25 The pharmaceutical compositions containing the active ingredients may suitably be formulated 25 so that they provide doses within these ranges either as single dosage units or as multiple dosage units.

Thus, it has been found according to the invention that the compounds of the formula I and the physiologically acceptable salts thereof can be used to inhibit herpesvirus multiplication. The 30 compounds of the formula I and physiologically acceptable salts thereof are useful in therapeutic 30

and/or prophylactic treatment of virus infections.

A preferred aspect of the invention is the use of the compounds of the formula I or a physiologically acceptable salt thereof, in the treatment of herpesvirus infections.

Working Examples

35 The following examples illustrate the preparation of compounds according to the invention. 35

Example 1.9-(2-oxo-4-hydroxybutyl)guanine

4-(2-Amino-6-chloropurin-9-yl)-3-oxobutyl o-chlorobenzoate (18 mg) was dissolved in 1M hydrochloric acid (20 ml) and refluxed for three hours. The solution was made alkaline with diluted 40 ammoniumhydroxide and evaporated to dryness. The residue was purified by preparative HPLC on a 40 reverse phase column (/u Bondapack C18 methanol—water 1:3) followed by chromatography on a Biogel P-2 column eluted with water. The residue 5.4 mg was a white solid which gave a single peak on HPLC (same solvent system as above).

nmr (DMSO-d6); 53.1 (2H, triplet, —CH2—), 4.1 (2H, NCH2CO) 4.3 (2H, triplet, —CH2—), 6.6 45 (2H, singlet, NH2, purine) 7.7 (1H, singlet H-8, purine). 45

4-(2-Amino-6-chloropurin-9-yl)-3-oxobutyl o-chlorobenzoate used as a starting material was prepared as follows:

0

ch^cchoch-oh 2,| 2 2

II

0

GB 2 122 197 A

a) 3-Hydroxybutyl o-chlorobenzoate ch3chch2ch2oc oh

Butane-1,3-diol (1.0 g) was dissolved in pyridine (50 ml) and cooled to 0°C. Ortho-chlorobenzoylchloride (1.9 g) was added with stirring. The reaction mixture was kept at 0°C for 1 hour 5 and then at room temperature over night. The solution was poured with stirring into ice-water (~100 5 ml). After stirring for 1 5 min the aqueous mixture was extracted with chloroform. The combined chloroform extracts were washed with ice-cold 0.25 M aqueous sulfuric acid, saturated aqueous sodium hydrogen carbonate and water. The dried solution (sodium sulfate) was concentrated to a syrup and purified by silica gel column chromatography (toluene-ethyl acetate 1:1) to yield 10 chromatographically pure 3-hydroxy-butyl o-chlorobenzoate (1.92 g). TLC in the same solvent Rf:0.56. 10 NMR (CDCI3): 51.24 (3H, d, —CHa), 1.63—2.10 (2H, m, —CH2—), 3.69—4.23

(1H,m,— CH—),

I

OH

4.29—4.66 (2H, m, —CH2—), 7.13—7.52 (3H, m, aromatic protons), 7.63—7.92 (1H, m, aromatic proton).

15 b) 3-Oxobutyl o-chlorobenzoate 15

CI

ch cch ch oc —/()>

3, 2 2 1

0 .0

Pyridiniumchlorochromate adsorbed to aluminium oxide (33 g, 32.8 mmol) (Cr03 • C5H5N ■ HCI prepared according to Yu-Shia Cheng et al., Synthesis, March 1980) was added to 3-hydroxybutyl o-chlorobenzoate (1.8 g, 7.8 mmol; prepared according to a)) in n-hexane (100 ml). After stirring at room 20 temperature over night the solid was filtered and the filtrate evaporated to dryness to give 1.21 g of 3- 20 oxobutyl o-chlorobenzoate. TLC toluene-ethyl acetate 1:1, Rf 0.74.

NMR (CDCI3): 52.21 (3H, s CH3), 2.81 (2H, triplet —CH2—), 4.54 (2H, triplet —CH—), 7.0— 7.45 (3H, m, aromatic protons), 7.45—7.84 (1H, m, aromatic proton).

c) 4-Bromo-3-oxobutyl o-chlorobenzoate

25 Br-CV|C¥H2°5"A^/ 25

0 0

3-Oxobutyl o-chlorobenzoate (670 mg, 2.96 mmol; prepared according to b)) was dissolved in anhydrous methanol (10 ml). The solution was stirred and cooled in an icebath, and bromine (0.15 ml, 2.96 mmol) was added rapidly. The reaction mixture was kept at +5°C for 18 h. Water (7 ml) and concentrated sulfuric acid (0.6 mi) were added and the mixture was stirred at room temperature over

30 night. 30

Water (10 ml) was added to the solution and the aqueous mixture was extracted with chloroform. The combined chloroform extracts were washed with saturated aqueous sodium hydrogen carbonate and water. The dried solution (sodium sulfate) was concentrated to a syrup and purified by silica gel column chromatography (toluene-ethyl acetate 16:1) to yield pure 4-bromo-3-oxobutyl o-35 chlorobenzoate. TLC in the same solvent, Rf:0.40. 35

NMR (CDCI3): 5 3.12 (2H, triplet —CH2—), 3.87

(2H, singlet Br—CH2—C),

II

0

4.57 (2H, triplet —CH2—), 7.12—7.48 (3H, m, aromatic protons), 7.48—7.81 (1H, aromatic proton).

10

GB 2 122 197 A 10

d) 4-{2-Amino-6-chloropurin-9-yl)-3-oxobutyl o-chlorobenzoate

CI

h2n

N

CI

ch2cch2ch2oc

2-Amino-6-chloropurine (36.6 mg, 0.216 mmol) and anhydrous potassium carbonate (30.0 mg, 0.216 mmol) were mixed with dimethylformamide (7 ml). The mixture was cooled in an icebath and 4-5 bromo-3-oxobutyl o-chlorobenzoate (66 mg, 0.216 mmol; prepared according to c)) in 5

dimethylformamide (1 ml) was added. After stirring at room temperature for 18 h, the mixture was filtered and the filtrate was evaporated to dryness. The residue was purified by preparative silica gel layer chromatography (chloroform-methanol 6:1) to yield pure 4-(2-amino-6-chloropurin-9-yl)-3-oxobutyl o-chlorobenzoate (18 mg).

10 TLC in the same solvent, Rf:0.40. 10

NMR (DMS0-d6): 53.15 (2H, triplet, —CH2—), 4.26 (2H, triplet, —CH2—), 5.09 (2H, singlet N— CH2—CO), 6.96 (2H, s. NH2 purine residue), 7.43—7.63 (3H, m, aromatic protons), 7.83—7.95 (1H, m, aromatic proton), 8.06 (1H, s, H-8, purine).

Example 2. 9-(2-oxo-3,4-dihydroxybutyl)guanine

0

,JL/^

15 M

\ 9

2 \ « 15

ch,cchch,oh 2 | 2

oh

A solution of 4-(2-amino-6-chloropurin-9-yl)-3-keto-01,02-isopropylidenebutane-1,2-diol (10 mg) in 1 ml of 70% aqueous formic acid was kept at 50°C for 18 h, evaporated to dryness, neutralized with aqueous ammonia and evaporated again. Preparative HPLC (C18 RP column, methanol-water 10:90) afforded pure 9-(2-oxo-3,4-dihydroxybutyl)guanine. 'H NMR (DMS0-de): 53.67 (d, 2H), C//20H; 20 4.18 (t, 1H) CA/OH; 5.10 (d, 2H) NCH2C0; 6.25 (broad s, 2H) H2N; 7.5 (s, 1H) H8. 20

UV (0.01 M hydrochloric acid): Jlmax (nm) 252, ~274 (infl.)

(H20, pH 7): 250, ~270 (infl.)

(0.01 M sodium hydroxide): 267, ~255 (infl.).

4-(2-Amino-6-chloropurin-9-yl)-3-keto-01,02-isopropylidenebutane-1,2-diol used as a starting 25 material was prepared as follows: 25

a) 4-Chloroacetyl-2,2-dimethyldioxolane ch3 ch3 °\ V ?

ch^chcch2c1

A solution of glyceroyl chloride acetonide [0.22 g, 1.32 mmol; T. Rechstein, A. Pedolin and A.

Grussner, Helv. Chim. Acta 18, 598 (1935)] in 2 ml of diethyl ether was added with stirring to a 30 solution of diazomethane (2.7 mmol) in 10 ml of diethyl ether. After 1 h a solution of hydrogen chloride 30 in diethyl ether (1 ml, 3.5 M) was added with stirring at room temperature, the solution was neutralized and dried with solid potassium carbonate and evaporated in vacuum to a small volume. The light yellow oil (146 mg, 62%) was used directly in the following step.

1H NMR (CDCL): 51.40 and 1.52 (2s, 2x3H) C(CH,),; 3.8—4.8 (m, 3H) CHCH,; 4.48 (s, 2H), 35 CH2CI. 35

GB 2 122 197 A 11

b) 4-{2-Amino-6-chloropurin-9-yl)-3-keto-01,02-isopropylidenebutane-1,2-diol

CI

ch,cchch,

2l \2

0 0

X.

CH3 CH3

A mixture of 2-amino-6-chloropurine (277 mg, 1.635 mmol), finely ground, anhydrous potassium carbonate (226 mg, 1.635 mmol) and 20 ml of dry dimethylformamide was warmed to 100°C for 5 5 min, then cooled, finally in ice-water. A solution of 4-chloroacetyl-2,2-dimethyldioxolane (146 mg, 5 0.817 mmol; prepared according to a)) in 5 ml of DMF was added dropwise with stirring at 0°C and the mixture was stirred at ambient temperature for 18 h. The solvent was evaporated in vacuum. Chromatography (150 g of silica gel, CHCI3-MeOH 9:1 by volume) gave 31 mg (14%) of 4-(2-amino-6-chloropurin-g-yD-S-keto-OVOMsopropylidenebutane-l^-diol.

10 1H NMR (CDCI3): 1.45 and 1.62 (2s, 2x3H) C(CH3)2; 4.2—4.4 and 4.67 (ABX system, Jvic 3Hz 10 and 7Hz) CHZCH; 5.15 (broad s, 2H) H2N; 5.18 (s,2H) NCH2; 7.77 (s, 1H) H8.

The following examples illustrate the preparation of pharmaceutical compositions of the invention. The wording "active substance" denotes a compound according to the present invention or a salt thereof.

15 Tablets ^ 5

Each tablet contains:

Active substance 20.0 mg

Maize starch 25.0 mg

Lactose 190.0 mg

20 Gelatin 1 -5 mg 20

Talc 12.0 mg

Magnesium stearate 1 -5 mg

250.0 mg

Suppositories

25 Each suppository contains: 25

Active substance 20.0 mg

Ascorbyl palmitate 1 -0 mg

Suppository base (Imhausen H or Witepsol® H) ad 2000.0 mg

Syrup

30 Active substance 0.200 g 30

Liquid glucose 30.0 g

Sucrose 50.0 g

Ascorbic acid 0.1 g

Sodium pyrosulfite 0.01 g

35 Disodium edetate 0.01 g 35

Orange essence 0.025 g

Certified colour 0.015 g

Purified water ad 100.0 g

Injection solution

40 Active substance 3.000 mg 40

Sodium pyrosulfite 0.500 mg

Disodium edetate 0.100 mg

Sodium chloride 8.500 mg

Sterile water for injection ad 1 -00 ml

12

GB 2 122 197 A 12

Sublingual tablets

Active substance

Lactose

Talc

Agar

Jelly

10

Active substance Methocel®

Methyl paraoxybenzoate Propyl paraoxybenzoate Sodium hydroxide and hydrochloric acid to pH 6.7 Distilled water ad

5.0 mg 85.0 mg 5.0 mg 5.0 mg

100.0 mg

1-0 g 4.0 g 0.12 g 0.05 g-

100.0 ml

10

15 Ointment I

20

Active substance Cetyltrimethylammoniumbromide Stearyl alcohol Cetanol

Liquid paraffine Glycerol

Hydrochloric acid to pH 6.5 Distilled water ad

1.0 g 0.6 g 2.25 g 6.75 g 17.0 g 12.0 g

100.0 g

15

20

Ointment II

25

Ointment III

30

Active substance Polyethylene glycol 1500 Polyethylene glycol 4000 Propylene glycol ad

Active substance Sorbitan monoleate Petrolatum ad

3.0 g 50.0 g 15.0 g 100.0 g

3.0 g 5.0 g 100.0 g

25

30

Ointment IV

35

40

Ointment V

45

50

Eye drops I

55

Active substance

Adepslanae

Tween® 60

Span® 40

Paraffin, liquid

Propylene glycol

Hydrochloric acid to pH 6.5—8

Sterile water ad

Active substance Adepslanae Tween® 60 Span® 40 Paraffin, liquid Propylene glycol Boric acid

Sodium hydroxide to pH 6.5—8 Sterile water ad

Active substance Disodium edetate Sodium chloride for isotonia q.s. Hydrochloric acid to pH 6.5—8 Methocel® 65 HG 4000 Sterile water ad.

5.0 g 20.0 g 4.0 g 2.0 g 4.0 g 5.0 g

100.0 g

5.0 g 20.0 g 4.0 g 2.0 g 4.0 g 5.0 g 2.0 g

100.0 g

0.1 g 0.10 g

0.65 g 100.0 ml

35

40

45

50

55

13

GB 2 122 197 A 13

Eye drops II

Active substance Disodium edetate Sodium chloride for isotonia q.s. Hydrochloric acid to pH 6.5—8 Methocel® 65 HG 4000 Sterile water ad

0.3 g 0.10 g

0.65 g 100.0 ml

Eye drops III

10

Active substance Disodium edetate Sodium chloride for isotonia q.s. Boric acid

Methocel® 65 HG 4000 Sterile water ad

0.2 g 0.1 g

0.1 g 0.65 g 100.0 ml

10

15 Eye ointment I

Active substance Paraffin oil Petrolatum

3.0 g 19.0 g 78.0 g

15

Cream

20

25

30

Active substance Arlaton®

Cetanol Stearic acid Paraffin oil Propylene glycol Glycerol

Methyl p-hydroxybenzoate Propyl p-hydroxybenzoate Sodium hydroxide

Hydrochloric acid 2M to pH 8.0 (water phase Distilled water to

3.0 g 4.0 g 2.0 g 2.0 g 2.0 g 2.0 g 1.5 g 0.06 g 0.03 g 0.002 g

100.0 g

20

25

30

Jelly

35

40

Active substance Methocel®

Glycerol Tween®

Methyl p-hydroxybenzoate Propyl p-hydroxybenzoate Sodium hydroxide Hydrochloric acid 2M to pH 8.0 Distilled water to

3.0 g 2.45 g 10.0 g 0.1 Og 0.06 g 0.03 g 0.002 g

100.0 g

35

40

45

Tablets

Each tablet contains:

Active substance

Starch

Lactose

Polyvinylpyrrolidone Magnesium stearate

50

55

100.0 mg 60.0 mg 190.0 mg 5.0 mg 5.0 mg

360.0 mg

45

Biological tests

The inhibiting effect of compounds of the invention on herpesvirus was tested using the method described below. The cellular toxicity of the compounds on host cell functions was also tested. The following compounds were tested:

9-(2-oxo-4-hydroxybutyl)guanine VIS 590 9-(2-oxo-3,4-dihydroxybutyl)guanine VSA 687

I. Inhibition of virus multiplication in cell cultures

The inhibition of herpesvirus by VIS 590 and VSA 687 has been measured as plaque reduction according to the following procedure.

50

55

14

GB 2 122 197 A 14

The plaque reduction assay for herpes simplex type 1 was performed on Vera (Green Monkey Kidney) cells as described by Ejercito et al.( J. Gen. Virol. 2 (1968) 357. Monolayers on 5 cm petri dishes were used and after virus adsorption the compounds to be tested were added to the medium. The results are shown in table 1.

5 Table 1

Inhibition of herpes simplex type 1 plaque on Vero cell monolayers

Cone.

Test

Inhibition fiM

compound

%

10

VIS 590

35

100

VIS 590

>90

35

VSA 687

>90

10 100 VIS 590 >90 10

II. Cellular toxicity

VIS 590 was tested for cellular toxicity as shown in Table 2. The method used has been described by Stenberg (Biochemical Pharmacology, Vol. 30 (1980), 1005—1008). It is seen that VIS 15 590 did not affect cell growth at 100 /uM. However, at this concentration, herpes virus multiplication 15 was inhibited to more than 90% (see Table 1).

Table 2

Cellular toxicity of VIS 590 expressed as percent reduction in cell growth after 48 h of incubation

20

Percent reduction in

Cone.

Test growth of Vero cells.

pM

compound determined as cell number

100

VIS 590

0

20

Claims (1)

1. Claims

   25 1. A compound of the formula 25

   h2n n

   I

   • ch - ch-oh l 2

   0 R1

   wherein R, is hydrogen, hydroxy or mercapto; and physiologically acceptable salts or optical isomers thereof.

   2. A compound according to Claim 1, which is 9-(2-oxo-4-hydroxybutyl)quanine; and

   30 physiologically acceptable salts thereof. _ 30

   3. A compound according to claim 1, which is 9-(2-oxo-3,4-dihydroxybutyl)guanine; and physiologically acceptable salts or optical isomers thereof.

   4. A compound according to claim 1, which is 9-(2-oxo-3-mercapto-4-hydroxybutyl)guanine; and physiologically acceptable salts or optical isomers thereof.

   35 5. A pharmaceutical composition comprising as an active ingredient a compound or salt or 35

   isomer according to any one of the preceding claims.

   6. A pharmaceutical composition according to claim 5 designed for systemic administration.

   7. A pharmaceutical composition according to claim 5 designed for topical administration.

   8. A pharmaceutical composition according to claim 5 substantially as hereinbefore described

   40 with reference to any one of the Examples. 40

   9. A method for the preparation of a compound of formula I as defined in claim 1 comprising condensing an acyclic side chain of the formula

   15

   GB 2 122 197 A 15

   X €H2 C CH CH20R4

   /\ I

   R2 R3 R5 to the N-9 position of a guanine derivative of the formula followed by removal of any protecting groups, in which formulas X is a group such as chlorine,

   5 bromine, iodine or a group 0S02R9 where R9 is alkyl containing 1—8 carbon atoms, fluorinated alkyl 5 containing 1 —8 carbon atoms, aryl or alkylaryl, Y is hydrogen or a quaternary ammonium ion; R2 and R3 are together double-bonded oxygen, or alkoxy groups forming open or cyclic ketals; R4 is hydrogen or a hydroxyl protecting group; Rs is R, as defined above; R4 and R5 may optionally together form an epoxide when R, is OH, or R4 and Rs may optionally form a cyclic derivative such as a carbonate ester, 10 carbonate thioester or the corresponding ortho acid cyclic derivatives or cyclic acetal type compounds; 10 R6 is hydroxyl, chlorine, bromine, iodine, thiol, thioether, S02R9 where Rg is as defined above; or an oxygen derivative OR,, where R,, is alkyl, arylalkyl such as benzyl, substituted silyl, phosphoryl diester, phosphinothioyl, or S02R9 where R9 is as defined above; R7 and R8 are the same or different and are hydrogen or R10 where R10 is as defined above.

   15 10. A method for the preparation of a compound of formula I as defined in claim 1 comprising 15

   substituting groups in a compound of the formula

   OH

   / \

   ■CH— I

   R12

   ■ ci^OR^

   followed by removal of possible protecting groups, in which formula R2, R3, R4, R7 and R8 are as defined in claim 9 and R,2 is iodine or 0S02R9 where R9 is as defined in claim 9.

   20 11. A method for the preparation of a compound of formula I as defined in claim 1 comprising reducing the ester group to an alcohol in a compound of the formula co2R9

   followed by removal of any protecting groups, in which formula R2, R3, R5, R6, R7, R8 and Rg are as defined in claim 9.

   25 12, A method for the prepararation of a compound of formula I as defined in claim 1 comprising 25

   ring closure of a compound of the formula

   16

   GB 2 122 197 A

   16

   CH-jOR^

   followed by removal of any protecting groups, in which formula R2, R3, R4 and R5 are as defined in claim 9, Z is NH2 or an alkoxy group; and R13 is NH2 or guanidine.

   13. A method for the preparation of a compound of formula I as defined in claim 1 comprising ring closure of a compound of the formula h2N

   V

   K-

   hn

   AX

   Rl4

   nh

   I

   CH^"

   R2NR3

   ■CH2OR^

   followed by removal of any protecting groups, in which formula R2, R3, R4 and R5 are as defined in claim 9, and R14 is nitroso, nitro, amino, formic amide or amino ortho ester;

   14. A method for the preparation of a compound of formula I as defined in claim 1 comprising 10 substitution in the pyrimidine ring of a compound of the formula 10

   CJ^OR^

   followed by removal of any protecting groups, in which formula R2, R3, R4 and Rs are as defined in claim 9, Hal is fluoro, chloro, bromo, or iodine, and R15 is hydroxyl or amino, whereby when R15 is amino the amino group is converted to a hydroxyl function;

   15 15. A method according to any one of claims 9 to 14 for the preparation of a compound 15

   according to any one of claims 2 to 4.

   16. A method according to any one of claims 9 to 15, wherein a base obtained is converted to a pharmaceutically acceptable salt or a salt obtained is converted to the base or to a different, pharmaceutically acceptable salt, and/or an isomeric mixture obtained is separated into a pure

   20 enantiomeric isomer. 20

   17. A method according to any one of claims 9 to 16 substantially as hereinbefore described with reference to any one of the Examples.

   18. A compound or salt or isomer according to any one of claims 1 to 4 or a composition according to any one of claims 5 to 8 for use in a method of treatment of the human or animal body by

   25 surgery or therapy or of diagnosis practised on the human or animal body. 25

   19. A compound, salt, isomer or composition according to claim 18 for use in the treatment of virus infections.

   20. A compound, salt, isomer or composition according to claim 19 for use in the treatment of herpes virus infections.

   30 21. A compound, salt, isomer or composition according to claim 18 for use in the treatment of 30 neoplastic diseases.

   Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1984. Published by the Patent Office, 25 Southampton Buildings, London, WC2A 1 AY, from which copies may be obtained.